5
The basics of selecting fluorophores Choose the brightest fluorophores that can be used on your instrument Use brightest fluorophore for your least expressed protein Use the dimmest fluorophore for your most highly expressed protein (CD45, CD8, CD3) Avoid spillover from bright cell populations into detectors requiring high sensitvity Spectral viewer tools Use tandems with caution Essential for large multicolor panels Susceptible to degradation by light exposure or fixation Prevent light exposure Use appropriate fixation buffers and protocols Avoid acidic buffer conditions with FITC labeled samples because FITC is sensitive to low pH Avoid exposing samples and reagents to bright light Avoid incubating cells in fixative for extended periods of time, as this may affect fluorescence, particularly of tandems dyes Acquire samples with 6 hours of staining